GDNF缺失突变体的设计及其结构与功能关系(英文)

研究了GDNF结构与功能的关系 .基于鼠源GDNF的晶体结构 ,利用计算机SGIIndigo2(R4 4 0 0 )工作站和InsightⅡ (95.5)蛋白质分析软件模拟了人GDNF三维结构 ,设计了GDNF分子的两个缺失突变体ΔN1 2 8和ΔN78 90 .以野生型GDNFcDNA作为模板 ,用PCR法得到编码缺失突变体的DNA片段 .将大肠杆菌作为表达系统 ,使缺失突变体GDNF在大肠杆菌中表达 ,对表达产物纯化和复性后进行生物活性测定 .两株突变体在大肠肝菌中获得了高效表达 ,纯化后的GDNF突变体ΔN1 2 8可以与存在于KG 1a细胞表面的受体结合 ,但不能促进 8日龄鸡胚背根节突起的生长 .突变体ΔN78 90既不能与受体结合 ,同时也失去了促背根节突起生长的功能 .说明GDNF分子的N端氨基酸对分子的生物学活性很重要 ,但对分子与受体GDNFR α的结合并不是必需的 ,而分子中的螺旋区对分子与受体的结合以及生物学活性都必不可少 .

Design for GDNF Mutants and the Relationship between Its Structure and Function

To explore the relationship between the structure and function of GDNF,on the basis of the X ray crystal diffraction structure of rat GDNF,the tertiary structure of human GDNF molecule was modeled by the web site of SGI Indigo 2(R4400).Two mutants of GDNF were designed that the first one was for the study on the biological activity of its N terminal domain.The mutant was constructed by deletion of the 1st to the 28th amino acid residues of N terminus and termed as mutant △N1 28. The 2nd mutant was obtained by deletion of the 78th to the 90th amino acids residues of GDNF,which was a helix domain of GDNF structure,and termed as mutant △N78 90.The mutant DNAs were obtained by PCR amplification.The mutant GDNFs were expressed in E.coli. The biological activities of mutant GDNFs were elucidated by biological assay.Two mutant GDNFs were efficiently expressed in E.coli. The purified GDNF mutant △N1 28 binded to the GDNF receptor on KG 1a cell surface,but could not promote the neurite outgrowth of chicken embryo (8 days) DRG (dorsal root ganglion).The mutant △N78 90 could neither bind to the GDNF receptor,nor promote the neurite outgrowth of chicken embryo DRG. The results show that The N terminal domain of GDNF molecule is essential for GDNF exerting its biological activity,but not necessary to interact with its receptor.The helix domain of GDNF is indispensable to both binding with receptor and producing biological activity.